Membrane TNFα Target Cells
Assess the Mechanism of Action of Biologics Designed to Affect TNFα Signaling
- Prequalified according to ICH guidelines
- 96- and 384-well plate formats
- No primary cell culture required
- Supplied in convenient thaw-and-use format; also available in cell propagation model (CPM) format
Catalog Number:
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Catalog Number: J3331
Catalog Number: J3335
Catalog Number: J3322
エフェクター機能と抗体結合親和性の測定
腫瘍壊死因子α（TNFα）は、免疫反応や炎症反応に関与する重要なサイトカインです。TNFα は主にマクロファージによって産生されますが、他の白血球や内皮細胞、心筋細胞などでも産生されます。TNFαは、その受容体であるTNF受容体1型（TNFR1）およびTNFR2に結合することで、細胞の状況に応じた無数の細胞応答を誘導することができます。典型的には、TNFα が TNFR1 または TNFR2 に結合すると、免疫細胞の活性化、エフェクター機能の促進、サイトカインの産生などの炎症性反応や、急性期反応が引き起こされます。TNFαは、三量体の膜結合型（mTNFα）と可溶性タンパク質の両方で存在します。TNFα を標的とした抗体をmTNFα発現細胞に結合させると、抗体依存性細胞媒介性細胞毒性（ADCC）や補体依存性細胞毒性（CDC）などのエフェクター機能が誘導され、mTNFα発現炎症細胞を破壊することができます。しかし、このような研究は、mTNFαを自然に発現するモデル細胞がないために妨げられているのが現状です。
mTNFα 標的細胞は、細胞表面に TNFα を安定的に発現するように遺伝子操作された細胞株です。mTNFα 標的細胞は、抗TNFα ブロッカーの ADCC や CDC などのエフェクター機能を測定するアッセイで標的細胞として使用できるように設計されています。また、mTNFα に対する抗体の結合親和性の測定にも使用できます。
Assay Components
Membrane TNFα Target Cells are provided in thaw-and-use format (Cat.# J3331, J3335). These cryopreserved cells are ready to be thawed for use in assays that measure the effector functions of anti-TNFα blockers.
Membrane TNFα Target Cells are also available in Cell Propagation Model (CPM) format (Cat.# J3322). These cryopreserved cells can be thawed, propagated and banked for long-term use.
Workflow
Standardized Reagents Improve Results
- Thaw-and-use format provides significant time and labor savings over traditional methods while reducing variability.
- Easily amenable to high-throughput workflows.
Assay Principle
mTNFα Target Cells used to measure ADCC with the ADCC Reporter Bioassay. Following engagement with the Fc region of a relevant antibody bound to a Target Cell, ADCC Bioassay Effector Cells expressing the FcγR will transduce intracellular signals, resulting in NFAT-mediated luciferase activity that can be easily quantified.
mTNFα Target Cells used to measure CDC with the CytoTox-Glo™ Assay. The assay measures dead-cell protease activity, and the amount of luminescence directly correlates with the percentage of cells undergoing cytotoxic stress.
Assay Performance Data
Accuracy and precision are critical to bioassays and the development of biologics. This assay was developed and prequalified according to ICH guidelines for accuracy and precision, giving it the performance characteristics required for many applications of a bioassay used for antibody drug discovery research and development.
Accuracy: Measure of how close results are to a reference or "target" value.
IntraLab Precision: Measure of repeatability from multiple assays of the same homogeneous sample under the same operating conditions over a short time (same analyst; same lab; same day).
Intermediate Precision: Measure of repeatability of multiple assays of the same homogeneous sample with different analysts or equipment or on different days.
Linearity: Ability of the assay to provide measured values that are proportional to the concentration of the sample.
The ADCC Reporter Bioassay using mTNFα Target Cells Shows Precision, Accuracy and Linearity.
| Parameter | Results | |
|---|---|---|
| Accuracy | % Expected Relative Potency | % Recovery |
| 50 | 49.1 | |
| 70 | 72.0 | |
| 100 | 100.9 | |
| 140 | 150.1 | |
| 200 | 213.4 | |
| Repeatability (% CV) | 100% (Reference) | 2.1 |
| Intermediate Precision (% CV) | 9.4 | |
| Linearity (r2) | 0.9991 | |
| Linearity (y = mx + b) | y = 1.099x – 6.024 | |
| A 50–200% simulated potency series of infliximab was analyzed in triplicate in three independent experiments performed on three days by two analysts using mTNFα Target Cells in the ADCC Reporter Bioassay. Bio-Glo™ Reagent was added and luminescence quantified using the GloMax® Discover System. Data were analyzed and relative potencies calculated after parallelism determination using JMP® software. Data were generated using thaw-and-use cells. | ||
The ADCC Reporter Bioassay with mTNFα Target Cells measures the activity of the anti-TNFα antibodies infliximab (Panel A) and adalimumab (Panel B). mTNFα Target Cells were added to a 96-well assay plate 18 hours prior to the assay. On the day of the assay, ADCC Effector Cells and a titration of infliximab or adalimumab were added. After a 24-hour induction at 37°C, Bio-Glo™ Luciferase Reagent was added and luminescence measured using the GloMax® Discover System. Data were fitted to a four-parameter logistic curve using GraphPad Prism® software. The EC50 was 148ng/ml for infliximab and 10.7ng/ml for adalimumab. Fold induction was 4.9 and 2.9 for infliximab and adalimumab, respectively.
A CDC assay measures the activity of the anti-TNFα antibodies infliximab (Panel A) and adalimumab (Panel B). mTNFα Target Cells were added to a 96-well assay plate 18 hours prior to the assay. On the day of assay, 10% CDC-Qualified Human Serum and a titration of infliximab or adalimumab were added. After a 6-hour induction at 37°C, CytoTox-Glo™ Cytotoxicity Reagent was added and luminescence measured using the GloMax® Discover System. Data were fitted to a four-parameter logistic curve using GraphPad Prism® software. Infliximab gave an EC50 of 0.73µg/ml, fold induction of 7.7 and specific lysis of 40.2%. With adalimumab, the EC50 was 0.19µg/ml, the fold induction was 15.7 and the specific lysis was 37.1%.
Specifications
Catalog Number:
選択製品の構成品内容
| Item | Part # | Size |
|---|---|---|
Membrane TNFα Target Cells |
J333A | 1 × 0.5ml |
SDS
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使用制限
Not For Medical Diagnostic Use.保存条件
Please see the Protocol for detailed storage conditions.
NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.
This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use; (2) discovery, development and monitoring of biologic drugs and vaccines; (3) quality assurance testing of biologic drugs and vaccines; and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. “Commercial use” means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to propagate, modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells. In addition, recipient must use luminescent assay reagents purchased from Promega for all luminescence-based assays using this product, or contact Promega to obtain a license for use of this product with reagents other than Promega’s. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.
選択製品の構成品内容
| Item | Part # | Size |
|---|---|---|
Membrane TNFα Target Cells |
J333A | 5 × 0.5ml |
SDS
Search for SDS分析証明書
使用制限
Not For Medical Diagnostic Use.保存条件
Please see the Protocol for detailed storage conditions.
NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.
This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use; (2) discovery, development and monitoring of biologic drugs and vaccines; (3) quality assurance testing of biologic drugs and vaccines; and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. “Commercial use” means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to propagate, modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells. In addition, recipient must use luminescent assay reagents purchased from Promega for all luminescence-based assays using this product, or contact Promega to obtain a license for use of this product with reagents other than Promega’s. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.
選択製品の構成品内容
| Item | Part # | Size |
|---|---|---|
Membrane TNFα Target Cells (CPM) |
J332A | 2 × 1ml |
SDS
Search for SDS分析証明書
使用制限
Not For Medical Diagnostic Use.保存条件
Please see the Protocol for detailed storage conditions.
NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.
This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use; (2) discovery, development and monitoring of biologic drugs and vaccines; (3) quality assurance testing of biologic drugs and vaccines; and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. “Commercial use” means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells except that recipient may propagate and store the cells for long-term use. In addition, recipient must use luminescent assay reagents purchased from Promega for all luminescence-based assays using this product, or contact Promega to obtain a license for use of this product with reagents other than Promega’s. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.
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